Our research focuses on the study of a fundamental membrane function of mammalian cells, namely the transport of macromolecules into cells through endocytosis. Because poly(lysine) is avidly taken up by endocytosis, it can be used as a carrier for other macromolecules or for drugs that are otherwise poorly transported. By conjugating a fragment of poly(lysine) of 9,000 Mr to horseradish peroxidase at a ratio of 1:1, the cellular uptake of active enzyme by cultured fibroblasts has been increased 1,000-fold. By conjugating methotrexate (MTX) to a poly(lysine) of 70,000 Mr at a ratio of 13:1, the cellular uptake of MTX is increased 20-\to 40-fold in normal CHO cells, and 200-\to 400-fold in CHO mutants defective in drug transport. In these cells, resistance due to defective MTX transport can be overcome by using MTX-poly(lysine), but not by using MTX-poly(D-lysine). This difference is due to the fact that D-isomer is not degraded and does not release active drug inside cells. These two conjugates are now used in two further lines of investigation, namely: (1) the isolation and characterization of mutants which are sensitive to MTX, but resistant to MTX-poly(lysine), and which are therefore defective in their ability to process a foreign macromolecule. Several such mutants have been found to be pleiotropically defective in endocytosis, and to show interesting patterns of cross resistance to plant and bacteriol toxins; and (2) the development of specific linkages between drugs and poly(D-lysine). Three different types of linkages were shown to restore the cytotoxicity of drug-X-poly(D-lysine), namely a tripeptide that is cleaved by proteolysis, an acid-sensitive linkage that cleaves in acidic endosomes-lysosomes, and a disulfide linkage that is reduced in a yet undefined compartment. Two other macromolecular carriers are being examined for their potential ability to target drugs to tumor cells. A methotrexate-carrying human serum albumin, complexed with anti-human serum albumin IgG, has been used to kill selectively two Fc-Receptor positive tumor cells lines in vitro. A MTX-carrying monoclonal anti-tumor antibody (anti-SSBA-1) is being used to kill tumor cells that carry the SSEA-1 antigen. (A)